Nucleocytoplasmic distribution and dynamics of the autophagosome marker EGFP-LC3.
Nucleocytoplasmic distribution and dynamics of the autophagosome marker EGFP-LC3.
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自噬体标记物 EGFP-LC3 的核质分布和动态。
DOI：
10.1371/journal.pone.0009806
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发表时间：
2010-03-23
期刊：
影响因子：
3.7
通讯作者：
Kenworthy AK
中科院分区：
文献类型：
--
作者：
Drake KR;Kang M;Kenworthy AK
自噬的过程涉及自噬小体的形成，自噬小体是包裹胞浆的双层结构。微管相关蛋白轻链3(Microtubule-Associated Protein Light Chain 3，LC3)是第一个在哺乳动物细胞中特异性标记自噬细胞膜的蛋白质，随后EGFP-LC3成为应用最广泛的自噬报告基因之一。虽然目前认为Lc3主要在自噬小体形成的胞浆中发挥作用，但在已发表的荧光图像中，EGFP-Lc3通常出现在相对于细胞质的核质中。然而，以前的报道中还没有对EGFP-LC3的核池进行专门的研究，并且目前还不清楚LC3穿梭于细胞质和核质之间的机制。因此，在这项研究中，我们研究了EGFP-LC3在活细胞中核质分布的调节。通过定量荧光显微镜分析，我们证明了在两个常用的研究细胞系COS-7和HeLa中，可溶性的EGFP-LC3确实相对于细胞质在细胞核中富含。虽然Lc3含有一个可能的核输出信号(NES)，但抑制活跃的核输出或NES的突变对EGFP-LC3的核质分布没有影响。此外，FRAP分析表明，在稳态条件下，EGFP-LC3经历了有限的被动核质转运，并且EGFP-LC3在细胞核和细胞质中的扩散迁移率比自由扩散单体的预测要慢得多。自噬的诱导导致与自噬结合蛋白相比，可溶性EGFP-LC3的水平明显下降，但对其余可溶池的核质比或扩散迁移率影响不大。我们的结论是，胞核中可溶的EGFP-LC3的浓缩独立于活跃的核输出或自噬的诱导。相反，将可溶性的EGFP-LC3掺入细胞质和细胞核内的大分子复合体可能会阻止其在两个隔室之间的快速平衡。
The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol. Microtubule-associated protein light chain 3 (LC3) was the first protein shown to specifically label autophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy. Although LC3 is currently thought to function primarily in the cytosol, the site of autophagosome formation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However, the nuclear pool of EGFP-LC3 has not been specifically studied in previous reports, and mechanisms by which LC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonly studied cell lines, COS-7 and HeLa. Although LC3 contains a putative nuclear export signal (NES), inhibition of active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore, FRAP analysis indicates that EGFP-LC3 undergoes limited passive nucleo-cytoplasmic transport under steady state conditions, and that the diffusional mobility of EGFP-LC3 was substantially slower in the nucleus and cytoplasm than predicted for a freely diffusing monomer. Induction of autophagy led to a visible decrease in levels of soluble EGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-cytoplasmic ratio or diffusional mobility of the remaining soluble pools of EGFP-LC3. We conclude that the enrichment of soluble EGFP-LC3 in the nucleus is maintained independently of active nuclear export or induction of autophagy. Instead, incorporation of soluble EGFP-LC3 into large macromolecular complexes within both the cytoplasm and nucleus may prevent its rapid equilibrium between the two compartments.
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影响因子：
8
作者：
Furuta, S;Miura, K;Kamata, T
通讯作者：
Kamata, T
影响因子：
13.3
作者：
Cieehomska, Iwona A.;Tolkovsky, Aviva M.
通讯作者：
Tolkovsky, Aviva M.
DOI：
10.1007/b102214
发表时间：
2005-01-01
期刊：
MICROSCOPY TECHNIQUES
影响因子：
--
作者：
Houtsmuller, AB
通讯作者：
Houtsmuller, AB
DOI：
10.1083/jcb.200412022
发表时间：
2005-05-09
期刊：
The Journal of cell biology
影响因子：
--
作者：
Komatsu M;Waguri S;Ueno T;Iwata J;Murata S;Tanida I;Ezaki J;Mizushima N;Ohsumi Y;Uchiyama Y;Kominami E;Tanaka K;Chiba T
通讯作者：
Chiba T
影响因子：
3.4
作者：
Kang, Minchul;Day, Charles A.;DiBenedetto, Emmanuele
通讯作者：
DiBenedetto, Emmanuele